Recently, it has been speculated that a 5-hydroxymethyl cytosine (hmC) plays a role in mammalian gene expression, specifically in embryonic stem cells and neuronal cell development as an intermediate step in demethylation (Tahiliani et al., Science 324(5929): 930-5 (2009); Kriaucionis and Heintz, Science 324(5929): 929-30 (2009)). A TET family of enzymes has been described that catalyzes the conversion of 5-methylated cytosine (mC) to hmC (WO 2010/037001). Detection of hydroxymethylation has proved challenging. Chemical methods, most commonly sodium bisulfite sequencing used to detect mC, do not discriminate between mC and hmC (Cokus et al., Nature 452: 215-219 (2008). PCT Publication No. WO 2010/037001 describes the use of antibodies that bind directly to hmC. A product that includes mixtures of endonucleases allows detection of hmC by subtraction (EpiMark™, New England Biolabs, Inc. (NEB) and PCT/US10/46632). This product utilizes T4 β-glucosyltransferase (BGT) to glucosylate the hmC which is then resistant to endonuclease cleavage permitting differentiation from mC. It would be desirable to develop simple rapid and direct methods to detect and analyze the presence of hmC in a polynucleotide sequence context which additionally are capable of discriminating hemi-hmC from symmetric hmC so as to precisely identify the cytosine (C) that is hydroxymethylated.